Histones

For cell culture experiments, polySia was purified as described above. 5B8 cells were cultivated and treated as described in detail earlier [39 (link)]. For the experiments, 30,000 cells per well were seeded on a 96-well plate. In addition to untreated cells, cells were incubated with histones (40 µg/mL; Sigma Aldrich) at 37 °C and 5% CO₂ for 1 h 45 min. In order to test the impact of polySia originating from plasma, native polySia- or endoN-treated polySia was added. The polySia of each well corresponded to the amount of polySia in 125 µL plasma. The cytotoxicity was determined by a lactate dehydrogenase (LDH) cytotoxicity assay (BioVision, Milpitas, CA, USA).

We monitored signal activity by quantifying expression of the smc2178::lacZ reporter fusion in a S. meliloti strain overexpressing the nsrA receptor protein (GMI12052) or an isogenic cyaK mutant derivative (S4 Table). A bacterial suspension (OD₆₀₀ = 0.1) was obtained by diluting an overnight culture of the reporter strains in synthetic modified Vincent medium [59 (link)] supplemented with gentamicin (20μg/ml) and tetracycline (10μg/ml). 950 μl of this suspension was mixed with 50 μl of the signal solution to be tested in a sterile polystyrene tube, incubated overnight at 28°C in a rotatory shaker (200 rpm). NCR peptides were assayed at the highest concentration (0.8 μM each) that did not impair bacterial growth, Histones at 0.33 μM and polymyxin B sulfate (Sigma Aldrich) at 0.36 μM. β-galactosidase activity was quantified as described before [16 (link)]. For specific activities assessment, the protein content of the assayed sample was quantified by the Bradford method (Bio-rad, USA). We adopted the following rule to illustrate statistical significance in figures: *, P < 0.05; **, P < 0.01; ***, P < 0.001; actual P-values are given in the text or in figure legends.